Assay devices which employ immunochromatographic principles are well known. Particularly common are “lateral flow” assay devices. In devices of the lateral flow type, a labelled specific binding reagent is releasably immobilised on a strip of porous material. A liquid sample is applied to one end of the porous strip and the capillary properties of the strip transports the liquid sample along the strip, releasing the labelled specific binding reagent, which binds specifically to the analyte of interest (at a first binding site thereof), if present, in the sample. The labelled binding reagent is then generally captured at a test zone by a second reagent having specific binding for a second binding site of the analyte of interest. Excess labelled binding reagent is then typically captured at a control zone, downstream of the test zone by a control reagent which binds specifically to the labelled reagent. Assay devices of this type are described in further detail in, for example EP 0 291 194.
In a known lateral flow assay device (of the sort disclosed in EP 0 291 194 and EP 0 560 411), such as the Clearblue® pregnancy test kit (available from Unipath Ltd, Bedford UK), which works by detecting the presence of hCG in a urine sample applied to the test device, two visible signals may be generated. One signal is a ‘control’ signal, and is formed by the localization of derivatised blue latex beads: the latex beads are coated with an immunoglobulin molecule and are captured by a capture antibody, deposited in a line on the test stick generally perpendicular to the direction of sample flow, the capture antibody having specific binding activity for the immunoglobulin carried on the beads. The generation of this signal informs the user that:    (i) neither the immunoglobulin on the latex bead, nor the capture antibody on the test stick, have been sufficiently denatured or otherwise degraded during manufacture or storage of the test kit significantly to interfere with the specific binding between the two molecules; and    (ii) sufficient liquid sample has been applied to mobilise the releasably immobilised latex beads and to transport them along the test stick at least as far as the “control” zone, in which the capture antibody is located.
The significance of (i) is that the immunoglobulin on the test stick is one which binds specifically to the analyte of interest. The control signal indicates that the capture antibody and the latex-bound antibody are still capable of associating, which implies that other immunoglobulin-based reagents (such as an analyte-specific immunoglobulin reagent) in the test kit should equally have retained their specific binding activity. The significance of (ii) is that the ‘control’ zone is located downstream of the ‘test zone’, in which the analyte is bound (together with any specifically-associated latex-labelled immunoglobulin) by a further analyte-specific immunoglobulin, deposited in a line on the test stick, generally perpendicular to the direction of flow of the sample. The line of immobilised analyte-specific immunoglobulin in the test zone is substantially parallel to, but upstream of, the line of immobilised antibody in the control zone.
In this way, a urine sample containing hCG contacted with the test stick in a correctly-performed assay, will cause the deposition of latex beads in both the control zone and in the test zone, resulting in the formation of two blue lines visible to the user, one line in the control zone and one line in the test zone.
The instructions provided with the kit direct the user to read the assay result 1 minute after removing the test stick from contact with the sample. Accordingly it is necessary for the user to have access to an external timer in order to read the assay result after the correct time interval has elapsed.
The label is generally a direct label, which is readily visible to the naked eye, so that a visual signal is generated wherever the labelled reagent accumulates in sufficient amount.
A problem with this type of assay device is that it takes a little while, after removing the assay device from contact with the liquid sample, for the test signal to appear. Clearly the user would like to read the result of the assay as soon as possible but, equally, the user requires confidence that sufficient time has elapsed for the proper assay result to have been obtained and that the test is not being read too early, without having to wait an inordinately long period.
In order to address this problem, it is known to incorporate a “timer” into an assay device, as described in EP 0 826 777. In particular, EP 0 826 777 discloses an assay device which comprises, in addition to the usual assay reagents, a variety of components which interact a pre-determined time interval after application of the sample to the test device, to create a detectable colour change. These additional ‘timer’ reagents are deposited in a ‘timer’ section of the test strip and, upon hydration by the sample, interact to produce a colour change.
In addition, the ‘timer’ reagents are also said to perform quality-control function. It is generally undesirable if assay devices are exposed to moisture. However, since the timer reagent when hydrated produces a coloured product, the timer will reveal if the device has been exposed to moisture. The ‘quality control’ function is however very limited, as it indicates only that the device has been exposed to an unquantified amount of moisture. The arrangement disclosed in EP 0 826 777 cannot indicate, for example, whether the test reagents have retained their specific binding properties, which can deteriorate with time, especially if the assay device has been stored in conditions of temperature, for instance, which are sub-optimal.